JP2000245483A - Transgenic plant body expressing antimicrobial peptide derived from seed of pharbitis nil l. - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new cDNA useful for producing a transgenic plant body expressing an antibacterial and an antifungal polypeptide, comprising a cDNA encoding an antimicrobial polypeptide derived from Pharbitis nil L. having a specific amino acid sequence. SOLUTION: This new cDNA encodes an antimicrobial peptide having an amino acid sequence of the formula (x is S or R; y is R or S; Z is S or nothing) and is useful for producing a transgenic plant expressing an antibacterial and an antifungal peptide derived from seed of Pharbitis nil L. and having resistance to pathogenic microorganisms. The cDNA is obtained by collecting a ripened seed of Pharbitis nil L. 40 days after flowering, extracting a poly(A) +RNA by a routine procedure, producing a cDNA library of plasmid base by using the poly(A)+RNA and screening the cDNA library by using a cDNA as a probe obtained by subjecting a cDNA derived from seed of Pharbitis nil L. with a primer composed of an amino acid sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to morning glory (Pharbitis).
nil L.) transgenic plants expressing antimicrobial and antifungal peptides derived from seeds. More specifically, the present invention relates to cDNA encoding an antimicrobial peptide derived from morning glory seeds, a recombinant plant expression vector of the peptide, and a transgenic plant having resistance to pathogenic microorganisms.

[0002]

BACKGROUND OF THE INVENTION Since plants do not have the motility that can be avoided from the invasion of pathogenic fungi, they must protect themselves by a direct or indirect response. From this point of view, pathogenic fungi (fungal pa
Control of thogen) has become an important issue, and efforts have been made to prevent fungal-caused diseases when cultivating crops. Primarily, previous studies for fungal control are:
Concentrated in the hygiene or chemical field, damage from fungal infections has been reduced to some extent with the development of effective fungicides. However, Botrytis
Develop crops that are resistant to a variety of fungi such as Botrytiscinerea, Fusarium oxysporum, Phytophthora parasitica, and Alternaria alterizata Breeding research and genetic engineering research could not achieve great results.

Recently, etiology-related (P
R) Dynamic defense system (dynam) for plants consisting of antibacterial agents belonging to different molecular weight groups such as protein (pathogenesis-related protein) and phytoalexin (phytoalexin)
ic defense system) has been studied. Plants do not have the immune system found in higher animals to protect themselves from pathogens, and they are able to protect themselves from invasion of pathogens, mainly by antibacterial agents such as various small antibacterial proteins rich in cysteine and glycine. Is protected.

[0004] Among such antibacterial proteins, chitin-binding proteins having a hevein region are known to share a similar sequence and exhibit various antibacterial activities, especially antifungal activities. For example, among chitin binding proteins, chitinase (see: Schumbaum, A. et al., Nature,
324: 365-367, 1986), wheat germ agglutinin (wheat germagg
lutinin, WGA, see: Andersen, NH et al., Biochem., 3
2: 1407-1422, 1993), barwin (see Hejgaa).
rd, J. et al., FEBS Lett., 307: 389-392, 1992), plantdefensin (see: Terras, FRG).
Et al., Plant Cell, 7: 573-588, 1995), lectin (see Pe).
umans, WJ et al., Plant Physiol., 109: 347-352, 199
5), an antimicrobial peptide derived from Mirabilis jalapa and Amaranthus caudatus (see: Broekaert, WF et al., Biochemistry, 31: 4)
308-4314, 1992; Cammue, BPA et al., J. Biol. Che
m., 267: 2228-2233, 1992).

[0005] It has not been elucidated to what extent chitin-binding proteins exhibit antifungal and / or antibacterial activity at the molecular level. Van Parijs et al., Heavenin and Uritica dio agglutinin
(ica agglutinin, UDA) has been reported to be associated with the size of its protein molecule (see: VanPa
rijs et al., Planta, 183: 258-264, 1991). That is, they predicted that the size of these proteins would be so small that they could cross the fungal cell wall to the plasma membrane, where they would affect the morphogenesis of the fungal cell wall. further,
In the case of thionin, a strongly basic, cysteine-rich antibacterial protein, it is speculated that thionin creates holes in the plasma membrane, disrupts the membrane, and results in the death of microorganisms. (See: Florak, DEA and Steikem
a, WJ, Plant Mol. Biol., 26: 25-37, 1994).

[0006]

By introducing a gene encoding an antifungal protein into a crop, infection with a fungal pathogen can be partially suppressed. Because it is based on specific pathogen recognition, the expressed protein of the transgenic plant does not show sufficient biological activity due to heat denaturation, and there is a risk of unexpected side effects from the plant Turned out to be unsatisfactory. Therefore, there has been a demand for the development of a technology that exhibits a wide range of antibacterial activity against various bacteria and fungi, and that can effectively increase the antibacterial activity of plants without causing any side effects.

[0007]

Therefore, the present inventors have made intensive research efforts to isolate an antimicrobial peptide that can impart an effective antibacterial activity to plants, is stable to heat, and has no side effects. As a result, mature morning glory (Pharbitis nil L.)
-AMP1 and Pn-AMP2 have stronger antibacterial activity than seeds
The peptides were separated and purified, and cDNAs encoding the above peptides were separated from each other by a cDNA library of morning glory seeds, and cloned into a plant expression vector. Next, a plant expressing the antimicrobial peptide was produced using Agrobacterium as a mediator, and it was found that the plant exhibited a wide range of antipathogenic activity, thereby completing the present invention.

Accordingly, a first object of the present invention is to provide cD encoding a novel antimicrobial peptide derived from mature morning glory seeds.
To provide NA. A second object of the present invention is to provide a plant expression vector containing the cDNA. A third object of the present invention is to provide a recombinant microorganism transformed with the expression vector and usable for producing a transgenic plant. A fourth object of the present invention is to provide a transgenic plant expressing the antimicrobial peptide. A fifth object of the present invention is to provide a method for producing a transgenic plant having anti-pathogenic bacterial resistance.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically. The antimicrobial peptide (hereinafter referred to as Pn-AMP) derived from mature morning glory seeds of the present invention is separated and purified from mature morning glory seeds and has the following amino acid sequence: QQCG-x-QA-y-GRLCGNGLCCSQWGYCGSTAAYCGAGCQSQCK-z (where x is S Or R, y represents R or S, and z is S
Or Pn-AMP1 and Pn-AMP2. Above, “Pn-AMP1” (the amino acid sequence is shown in SEQ ID NO: 1) is composed of 41 amino acids,
A peptide having serine, arginine and serine as the amino acids at positions 8 and 41, respectively, and “Pn
"-AMP2" (amino acid sequence is shown in SEQ ID NO: 2)
And a peptide having arginine and serine as the fifth and eighth amino acids, respectively.

Pn-AMP has a chitin-binding ability and a molecular weight of about 4
It is a basic peptide of kDa. Analysis of the amino acid sequence of Pn-AMP of the present invention revealed that Pn-AMP is a novel protein containing a cysteine- and glycine-rich chitin-binding domain with homology to other chitin-binding protein sequences. confirmed. In particular, the amino acid sequences of Pn-AMP1 and Pn-AMP2 showed 66% sequence homology to hevein, a rubber latex, and the sequence differences were frequently located in the variable region within the chitin-binding domain. Pn-AMP and other chitin
-In the binding protein, the cysteine residues were well conserved. Pn-AMP shows excellent antibacterial activity against a wide range of fungal species, regardless of whether the fungal cell wall contains chitin, and also shows antibacterial activity against some bacteria, but insects It did not show cytotoxicity against cells or mammalian cells.

Screening of a cDNA library of mature morning glory seeds to isolate the cDNA encoding the Pn-AMP revealed that Pn-A of about 609 bp and 605 bp respectively.
MP1 and Pn-AMP2 cDNA (SEQ ID NOs: 4 and 6) were selected. The Pn-AMP1 cDNA clone has a 5 ′ UTR (nucleotides 1-4).
6) consisting of Pn-AMP1 cDNA (nucleotides 47 to 325, SEQ ID NO: 5) and 3′UTR (nucleotides 326 to 609);
The Pn-AMP1 cDNA has an amino-terminal signal peptide region.
(Nucleotides 47 to 121), Pn-AMP1 (nucleotides 122 to 2)
44) and the carboxy-terminal region (nucleotides 245-325)
It was confirmed that it consisted of. On the other hand, Pn-AMP2 cDNA
Clones were 5 'UTR (nucleotides 1-21), Pn-AMP2 cD
NA (nucleotides 22 to 297, SEQ ID NO: 7), and 3′UTR
(Nucleotides 298-605), wherein the Pn-AMP2 cDNA
Is the amino terminal signal peptide region (nucleotides 22-9)
6), it was confirmed to be composed of Pn-AMP2 (nucleotides 97 to 216) and a carboxy terminal region (nucleotides 217 to 297). From these results, the Pn-AMP1 and Pn-AMP2 of the present invention were first expressed in the form of precursors consisting of 92 amino acids (SEQ ID NO: 8) and 91 amino acids (SEQ ID NO: 9), respectively. Through modification, it is suggested that it is converted to a mature peptide that exhibits antibacterial activity.

[0012] The present invention also provides a plant expression vector containing Pn-AMP cDNA which can be used for producing a fungal-resistant transgenic plant, and a transgenic plant transformed with the same. According to a preferred embodiment of the present invention,
A cDNA fragment containing a sequence corresponding to nucleotides 1 to 340 of Pn-AMP2 was cloned into a binary vector pGA643 to produce an expression vector pLES9806. As a result, Pn-AMP2 was expressed under the control of the CaMV 35S promoter. Next, Agrobacterium tumefaciens (Agrobacterium tum
efaciens) LBA4404 was transformed to obtain a transformant that can be used as a vehicle during the production of the transgenic plant, and the transformant was named `` Agrobacterium tumefaciens LBA4404 / pLES9806 '' and No. "KCTC 0548B" with the International Depository Agency, Korea Institute of Science and Technology (KIST), Institute of Biotechnology (KRIBB) Gene Bank (KCTC, 52, Ueong-dong, Yusung-gu, Daejeon, Republic of Korea)
P "as of November 18, 1998. Transgenic plants were produced using the microorganism as a vehicle.

Further, as an example of the antifungal-resistant transgenic plant expressing the Pn-AMP peptide of the present invention, the above-mentioned Agrobacterium tumefaciens LBA440
4 / pLES9806 (KCTC 0548BP) as a vector, Pn-AMP2 cDNA was added to tobacco leaf cells by leaf disc transformation.
Was transfected and cultured to produce a transgenic tobacco expressing Pn-AMP2. The transformed tobacco cells are referred to as "Nicotiana tabacum (Nicotiana tab
acum) cv. Xanthi-nc / pLES9806 ”and the International Depository Institute of Science and Technology (KIST) attached to the Institute of Biotechnology (KRIBB) Gene Bank (KCTC, Daejeon, Daejeon, Korea) No. 52, On-dong) under the deposit number "KCTC0549BP" on November 18, 1998.

In order to measure the effect of the introduced Pn-AMP expression on the degree of resistance of the transgenic plant to fungal infection, the transgenic tobacco was infected with Phytophthora parasitica pv. Nicotiana and cultured. Seven days after infection, disease symptoms began to be seen in wild-type tobacco, but in the case of transgenic tobacco, they showed no disease symptoms due to infection and were healthy. Fungal mycelium was spreading in leaves of wild-type plants, but no such mycelial growth was observed in leaves of transgenic plants. According to the present invention, resistance to fungal infection can be increased by introducing a gene encoding Pn-AMP into a target plant. Therefore, it is suggested that the recombinant expression vector of the present invention and the microorganism transformed thereby can be used for controlling fungal diseases of agricultural products.

[0015]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are exclusively for describing the present invention more specifically, and the scope of the present invention is not limited to these examples. It will be understood by those skilled in the art that the present invention is not limited to the example.

Example 1 : Isolation of antimicrobial peptides from mature morning glory seeds Example 1-1 : Isolation and purification of antimicrobial peptides 50 g of dried mature seeds of morning glory (Pharbitis nil L.) were turned into a fine powder. Pulverized, 10 mM NaH ₂ PO ₄ , 15 mM N
a ₂ Suspended in 500 ml of extraction buffer (pH 6.0) containing ₂ HPO ₄ , 100 mM KCl, 1 mM EDTA, and 1 mM thiourea. The suspension was stirred at 4 ° C. for 3 hours, and then centrifuged at 32,000 × g for 30 minutes to collect a supernatant. Solid ammonium sulfate was added to the supernatant to a concentration of 30% relative saturation. Next, the mixture was centrifuged at 32,000 × g for 30 minutes to remove the precipitate, and the supernatant was again adjusted to 70% relative saturation with ammonium sulfate.
The precipitate was obtained by re-centrifugation for 0 minutes. 180 ml of the precipitate
Was re-dissolved in deionized water and heat treated at 80 ° C. for 15 minutes to denature the high molecular weight proteins contained. Heat denatured protein at 32,000xg
Removed by centrifugation for 30 minutes. After the resulting supernatant was sufficiently dialyzed against deionized water, the solution was added to 10 mM phosphate buffer (pH 6.0) containing 25 mM NaCl, and CM5 equilibrated with the same buffer.
After injection into a 2-cellulose cation exchange column and washing several times, the protein adsorbed on the column was eluted with 10 mM phosphate buffer (pH 6.0) containing 520 mM NaCl, and the antibacterial activity of each fraction was examined. Fractions showing the activity of the CM-cellulose column were combined, and ODS-A12 was equilibrated with 10 mM phosphate buffer (pH 6.0).
0Reverse phase C ₁₈ open column (open column, 0.5cmx25cm, YM
C-gel, Japan), wash several times with water,
Elution was performed under the condition of a linear concentration gradient of acetonitrile up to 80%. Each fraction was examined for antimicrobial activity, and the active fractions were combined and dried in a vacuum concentrator. After dissolving the dried sample in 0.05% trifluoroacetic acid, an ODS-120T column (0.5 cm x 25 cm, Toso, Tok) was connected to a Gilson HPLC system.
yo, Japan). Solution A (0.05% (v / v) TFA / acetonitrile) with a linear gradient of 0-60%
TFA / water) was used to elute the protein within 60 minutes at a flow rate of 1 ml / min. As a result, two peaks appeared almost simultaneously on the elution curve, and the corresponding two fractions were collected respectively, subjected to reverse phase HPLC again under the same conditions as above, and further purified. As a result, the two peaks were correctly separated, and the single polypeptide contained in each peak had a molecular weight of about 4 kDa and exhibited excellent antibacterial activity against various fungi. -AMP1 "
And "Pn-AMP2".

Example 1-2 : Amino acid sequence analysis and chitin-
Characterization of the binding domain In order to determine the amino acid sequence of Pn-AMP1 and Pn-AMP2 purified in Example 1-1, each peptide was reduced and S-
Carboxymethylation and digestion with trypsin yielded peptide fragments suitable for sequencing (see: Creighton, JE
In Protein Structure, a practical approach, Creigh
ton, TE, pp155-167, IRL Press, Oxford, 1989).
The purified antimicrobial peptide (100-500 pmol) was dissolved in 50 ml of 0.4 M NH ₄ HCO ₃ containing 8 M urea, 5 ml of 45 mM DTT was immediately added, and the mixture was incubated at 50 ° C. for 15 minutes.
After cooling, 5 ml of a 100 mM iodoacetamide solution was added and the reaction mixture was incubated at room temperature for 15 minutes. Next, the mixture was diluted 3-fold with water, and the mixture was diluted with a reverse-phase HPLC column (Chemo).
cosorb 5-ODS-H, 2.1 mm x 100 mm). The peptide fragments were separated at a flow rate of 0.2 min / ml using 0-60% (v / v) linear gradient of acetonitrile. Each of the purified peptide fragments was subjected to ABI 473A Protein Sequencer (Applied Bio
amino acids sequences were analyzed using systems Inc., USA). As a result, mature Pn-AMP1 and Pn-AMP2 consisted of 41 and 40 amino acids, respectively, and were ranked 5th, 8th and 4th.
In the remaining portion except for the amino acid at position 1, it was confirmed that the two peptides share the same amino acid sequence. That is, Pn-AMP1 (SEQ ID NO: 1) is a peptide composed of 41 amino acids having serine as the 5th amino acid and arginine as the 8th amino acid, and having 41 serine as the 41st amino acid. -AMP2 (SEQ ID NO: 2) is a peptide consisting of 40 amino acids in which the fifth amino acid is arginine and the eighth amino acid is serine (FIG.
1). Therefore, it is assumed that Pn-AMP1 and Pn-AMP2 have a so-called isomer relationship, and Pn-AMP1 and Pn-AMP1
AMP2 was collectively referred to as "Pn-AMP".

On the other hand, analysis of sequence homology between the conventional chitin-binding protein and the Pn-AMP of the present invention revealed that Pn-AMP1
And Pn-AMP2 was confirmed to be a novel protein containing a chitin-binding domain rich in cysteine and glycine having homology to amino acids of the chitin-binding protein (FIG. 1). In FIG. 1, the sequences in the shaded boxes indicate portions of the same amino acid between the proteins, and the dots indicate the space introduced for optimal alignment. Hebein is rubber latex hebein, CBP20 is antifungal protein of tobacco, barley CHI is barley chitinase, rice CHI
Is a rice class 1 chitinase, UDA is Uritica dioica agglutinin, WGA3 is wheat germ agglutinin 3, isolectin, and Ac-AMP2 is an antifungal peptide derived from Amaranthus caudatus. Each is shown. The numbers on the right indicate amino acid numbers in each protein. As shown in FIG. 1, Pn-AMP1 and Pn-A
The amino acid sequence of MP2 is most similar to rubber latex hevein, showing 66% sequence homology, especially cysteine residues are well conserved in other chitin binding proteins from Pn-AMP1 and Pn-AMP2. Sequence differences are often found in the variable regions within the chitin binding domain. However, despite such high sequence homology, mature Pn-AMP1 and Pn-AMP2 (pI = 8.6) show stronger basicity than hevein (pI = 4.6), and this high pI value is Due to the presence of four arginine residues in AMP. On the other hand, as a result of measuring the chitin binding ability of Pn-AMP1 and Pn-AMP2 using a chitin column,
It was confirmed that both peptides had strong affinity for chitin.

Example 1-3 : Measurement of antibacterial activity of Pn-AMP The antibacterial activity of the antibacterial peptide separated and purified according to the present invention was determined by Br
Spectrophotometry (microspect) by oekaert et al.
rophotometry) (see: Broekaert, WF et al.,
Biochemistry, 31: 4308-4314, 1992). The test organisms were all eight plant-infecting fungi, including fungi without chitin in the cell wall, i.e., Botrytis ceria.
inerea), Colletotricum Randinarium
chum langenarium), Rhizoctonia solani (Rhizoctoni)
a solani), Fusarium oxysporum
sporum), Phytophthora parsica (Phytophthora par
asitica), Phytophthora caps
ici), Pythium spp. (Pythium spp.), and Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), along with the yeast Saccharomyces cerevisiae (Sac
charomyces cerevisiae) was used. All fungi are cultured on potato dextrose agar or V8 agar at 25 ° C., and mold spores or hyphae are harvested by the method of Duick et al.
uvick, JP et al., J. Biol. Chem., 267: 18814-18820, 19
92) and the standard method of Singleton et al. (See Singleton, LL).
Et al., In Methods for Research on Soil-borne Phytopat
hogenic Fungi, APS Press, Minnesota, 1992). Next, various concentrations of mature Pn-AMP1 and Pn-AMP2
The solution 20 [mu] l, 10 ⁴ pieces of mold spores per 1 ml, the growth medium (half strength containing 100 mold hyphae piece, or 10 ⁵ yeasts (h
alf-strength) potato dextrose broth) 80 μl
And in each well of a 96-well microplate. Here, sterilized distilled water was added to the control group instead of the peptide solution. After culturing at 25 ° C. for 48 hours, the growth inhibition rate is determined by measuring the absorbance at 595 nm. The concentration (IC ₅₀ ) was calculated. As a result, Pn-AMP1 and Pn-AMP2 show strong antibacterial activity against a wide range of plant-infectious molds and yeasts,
The IC ₅₀ for fungi is 0.6-75 μg / ml, which is the value of the amarant chitin-binding protein Ac-AM
Similar to IC _{50 of} P (see: Broekaert, WF et al., Bio
chemistry, 31: 4308-4314, 1992). Furthermore, Pn-AMP1 and P
n-AMP2 is not only a fungus that contains chitin, but also a fungus that does not contain chitin in the cell wall (Oomycetes)
It has a wide range of inhibitory effects, indicating that the chitin-binding ability of Pn-AMP is not essential for fungal growth inhibition.

On the other hand, thionine (see: Teras, FRG)
Al, Plant Physiol 103:. 1311-1319,1993) , such as Ac-AMP and plant defensins, some basic antimicrobial peptides that exhibit is reported to be strongly inhibited by cation, especially CaCl ₂ (See: Osborn, RW et al., FE
BS Lett. 368: 257-262, 1995). To examine the antagonistic effect of CaCl _{2 on} Pn-AMP1 and Pn-AMP2, the IC ₅₀ of the peptide was determined in the presence of 5 mM CaCl ₂ , and the IC ₅₀ value increased about 47- to 250-fold It was confirmed that.

To test whether Pn-AMP also has a growth inhibitory effect on gram-negative bacteria (E. coli, Agrobacterium tumefaciens) and gram-positive bacteria (Bacillus subtilis), these
The cells were cultured in the presence of Pn-AMP1 and Pn-AMP2. Mature Pn-AMP1 and Pn-AMP2 solution 20μl of various concentrations were mixed in each well of the LB medium 80μl and 96-well microplates containing 10 ⁵ bacteria per 1 ml. Next, the antibacterial activity of Pn-AMP was measured by IC ₅₀
As a result, even at a concentration of up to 200 μg / ml, there was no effect on the survival of Gram-negative bacteria. On the other hand, with respect to Bashir scan subtilis is a Gram-positive bacterium, in the case of Pn-AMP1 at an IC ₅₀ value of 38μg / ml, in the case of Pn-AMP2 was an IC ₅₀ value of 20 [mu] g / ml. In the presence of 5 mM CaCl ₂ , no growth inhibition was observed for both Pn-AMP1 and Pn-AMP2 at a concentration of 200 μg / ml.

[0022]Example 1-4: Pn-AMP cytotoxicity analysis In order to examine the cytotoxicity of the Pn-AMP of the present invention, monkey kidney
Cell line MA104 cell line and insect cell line Sf9
Spodoptera frungiperd
a) 9) Using the cell line, the method of Broekaert (Broekaert, W.
 F. et al., Biochemistry, 31: 4308-4314, 1992).
Cell viability test was performed: 1 × 10 ⁶Sf9 cells
And MA104 cells in Grace's media (Grace's media, Life
Technologies, Inc., U.S.A.), DMEM medium (Dulbecco's
odified Eagle medium, Life Technologies, Inc., U.
S.A). Next, in the presence of Pn-AMP1 and Pn-AMP2
Incubate further for 24 hours and stain with trypanblue
The cells were then assayed microscopically for viability. as a result,
Pn-AMP1 and Pn-AMP2 are used for animal cells (MA104) and insect cells.
Has no effect on the survival of the vesicle (Sf9)
Was.

Example 2 : Cloning of Pn-AMP cDNA Example 2-1 : Preparation of cDNA library derived from morning glory seed mRNA In order to clone cDNA encoding Pn-AMP, a cDNA library derived from morning glory seed mRNA was first used. Manufactured: 2 g of mature seeds from morning glory, 40 days after flowering, were used to extract total RNA and poly (A) + RNA by standard methods of Maniatis et al. Next, double-stranded (ds) cDNA was synthesized from 5 μg of the poly (A) + RNA using a λZAPII cDNA synthesis kit (Stratagene, USA) according to the manufacturer's instructions. 500 ng dscDNA to pBluescriptII (SK-) plasmid
(200 ng of Stratagene, USA) and ligated to the EcoRI / XhoI site to produce a plasmid-based cDNA library. Plasmid of the cDNA library was transformed into E. coli XL1-bl.
was introduced by electroporation into ue (Stratagene, USA), the results were cultured in LB medium containing 100 [mu] g / ml of ampicillin, approximately 10 ⁴ transformants was formed.

Example 2-2 : Selection of clone containing Pn-AMP cDNA A DNA probe for screening a cDNA library was prepared by using the ds cDNA prepared in Example 2-1 as a template and a polymerase chain reaction (PCR). PCR). At this time, as a primer, a CG1 primer having the following base sequence and synthesized by analogy with the amino acid sequence of the chitin binding domain found in Pn-AMP1 and Pn-AMP2 (CCSQWGYC): 5′-TG (C / T) TG (C / T) (A / T) (C / G) NCA (A / G) TGGGGNTA (C / T) TG
(C / T) GG-3 ′ (SEQ ID NO: 3) and an oligo (dT) primer were used.

The PCR consisted of 50 ng of ds cDNA, 100 ng of CG1 primer, 20 ng of oligo (dT) primer, Taq polymerase (Promega) and 10 × Taq polymerase buffer (Promeg
a) was performed in a final volume of 50 μl. The amplification program consists of an initial activation step at 94 ° C for 2 minutes followed by 30 ° C at 94 ° C.
The reaction was repeated for 40 cycles consisting of seconds, 50 ° C. for 30 seconds, and 72 ° C. for 1 minute, and finally an extension step at 72 ° C. for 10 minutes. After the reaction is completed, 1% of the amplified PCR product
Purify on agarose gel and use Random-Priming Labeling Kit: Amersh
am, USA) using α- ³² P [dCTP].

[0026] Example 2-3: Pn-AMP1 at about 10 ⁴ cDNA-containing plasmids were obtained by isolating Example 2-1 cDNA clones in Example 2-2 with respect to transformed E. coli colonies Manufactured
Using the PCR product labeled with ³² P, screening by indigo colony hybridization was performed.
As a result of the two screenings, 50 positive colonies were isolated. Plasmid DNA was extracted from each of the positive colonies, and the DNA was extracted with a Taq Dye Primer Cycle Sequencing Kit;
The nucleotide sequence of the ds cDNA was analyzed using an ABI373A automated DNA sequencer (Applied Biosystems Inc., USA) using in-Elmer, USA. 35 clones out of a total of 50 clones showed the same nucleotide sequence except that the lengths of the 5 'and 3' terminal portions were slightly different. The length of the longest cDNA clone among them is 609 bp (SEQ ID NO: 4). As a result of analyzing the nucleotide sequence by the DNASIS program, 5 ′ UTR (nucleotides 1 to 46) and Pn-AMP1 cDNA (nucleotide 47) were analyzed. 325; SEQ ID NO: 5), and 3 ′ UTR (nucleotides 326-609), and the Pn-AMP1 cDNA is a signal peptide region at the amino acid terminus (nucleotides 47-121), Pn-AMP1 (nucleotides 122-
244), and the carboxy terminal region (nucleotides 245-325)
It was confirmed that it consisted of.

Example 2-4 : Isolation of cDNA clone of Pn-AMP2 15 out of the 50 clones prepared in Example 2-3 except for the 35 clones described above were 5 'and 3''The same nucleotide sequence was shown except that the length of the terminal portion was slightly different. The length of the longest cDNA clone among them is 605 bp (SEQ ID NO: 6), but as a result of analyzing the nucleotide sequence by the DNASIS program, 5 ′ UTR (nucleotides 1 to 21), Pn-AMP2 cDNA (SEQ ID NO: 7) Pn-AMP2 cDNA consists of the amino-terminal signal peptide region (nucleotides 22-96), Pn-AMP2 (nucleotides 97-216), and carboxy. It was confirmed that it consisted of a terminal region (nucleotides 217 to 297). From these results, Pn-AMP1 and Pn-AMP2 of the present invention
92 amino acids (SEQ ID NO: 8) and 91 amino acids (SEQ ID NO:
It was suggested that after being expressed in the form of a precursor consisting of 9), it could be obtained as a mature peptide showing antibacterial activity by post-transcriptional modification.

Example 3 Construction of Pn-AMP2 Expression Vector and Production of Transformant In order to produce a Pn-AMP2 expression vector, a Pn-AMP2 cDNA was prepared.
PBluescriptIISK (-) vector containing XbaII / ClaI was completely digested with nucleotides 1-340 containing the entire coding region.
A 340 bp DNA fragment corresponding to the above was recovered on a 0.8% agarose gel. The 340 bp DNA fragment was converted into a binary vector pGA6.
The plant was ligated to 43 XbaII / ClaI sites to produce a plant expression vector pLES9806 (see FIG. 2). In FIG.
nptII represents a promoter and terminator of nopaline synthase, and a chimeric gene showing kanamycin resistance containing the coding region of neomycin phosphotransferase II, and CaMV 35S represents the cauliflower mosaic virus (CaMV) 35S promoter.

Next, after transforming Agrobacterium tumefaciens LBA4404 with pLES9806 by electroporation, 10 μg / ml kanamycin and 5 μg
Transformants were selected by plating on selective medium on LB agar plates containing / ml tetracycline and culturing at 28 ° C for 2 days. The selected transformant was named "Agrobacterium tumefaciens LBA4404 / pLES9806" and
Deposit No. on November 18, 1998 with the International Depository Research Institute of Biotechnology (KRIBB) Gene Bank (KCTC, 52 Uon-dong, Yongseong-gu, Daejeon, Korea) attached to the Korea Institute of Science and Technology (KIST) Deposited as "KCTC 0548BP".

Example 4 Production of Transgenic Tobacco Agrobacterium-mediated leaf disc transformatio
(n) was used to produce transgenic tobacco expressing Pn-AMP2 (see Horsch, R. et al., 1984, Science,
233: 496-498): Tobacco (Nicotiana tabacum cv.Xanthi-n
The leaf disk of c) was inoculated with Agrobacterium tumefaciens LBA4404 / pLES9806 (KCTC 0548BP) obtained in Example 3 and cultured for 2 days. Then, a selective medium (0.5 mg / l bovine serum albumin, Murashige-Skoog) was used. )
Salt, 3% sucrose, 100mg / l kanamycin, 250mg / l
(Including pseudopen and 0.8% agarose) to induce callus growth. After one month of culturing the callus, the regenerated shoots were transferred to a nursery bed where vermiculite, perlite and peat moss were mixed in the same ratio, and the day-length (day-length) in a 25 ° C incubator (day-length) 16 hours And grown. The obtained tobacco cells are referred to as "Nicotiana tabacum".
cv. Xanthi-nc / pLES9806 ”, on November 18, 1998, the Institute of Biotechnology (KRIBB) Gene Bank (KCTC, Ota, Korea) Deposit No. “KCTC 0”
549BP ".

Example 5 : P in transgenic tobacco
Investigation of n-AMP2 expression and antifungal resistance Example 5-1 : Immunoblot analysis The transgenic tobacco produced in Example 4 (KCTC 0
(549BP) was pulverized and put into an extraction buffer (50 mM sodium phosphate buffer, pH 6.0, containing 0.1 mM PMSF). The extract is centrifuged at 12,000 xg for 30 minutes, and the supernatant is
For 15 minutes. The heat denatured product was removed by centrifugation, and about 12 μg of a soluble protein was obtained through a concentration process, which was developed by 17% SDS-PAGE. The protein was transferred onto a nitrocellulose membrane filter by electroblotting, and the filter was washed with TTBS containing 6% (w / v) skim milk (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1%).
% Tween 20). The filters were then immunoblotted using rabbit anti-Pn-AMP2 antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G as secondary antibodies. ECL detection solution (Am
ersham, USA) to confirm that the Pn-AMP2 protein was expressed in the transgenic tobacco of the present invention. On the other hand, rabbit anti-Pn-AMP2 polyclonal antibody was prepared by immunizing rabbits with 1 mg of purified Pn-AMP2 bound to a silica matrix in Freund's complete adjuvant by subcutaneous injection, and immunizing 1 mg of the same antigen in Freund's incomplete adjuvant.
Were manufactured by boosting injection every 3 weeks.

Example 5-2 : Resistance of transgenic tobacco to fungal infection In Example 5-1 it was confirmed that the transgenic tobacco of the present invention expresses a Pn-AMP2 peptide having antibacterial activity. The antifungal properties of the transgenic tobacco were investigated by a simple method: Phytophthora parasitica pv. Nicotianae my on a V8 agar medium containing 1 g of dried normal tobacco leaves per liter.
celia) and cultured at 25 ° C. while exposing to light.
After the fungal mycelial flora has formed (2-4 days), agar disks (〜8 mm ² ) containing the mycelium are cut out and transgenic or non-transgenic tobacco plants are harvested for 4 days.
The cells were transferred to a weekly cultured MS agar medium. These were cultured in an incubator at 25 ° C. with a day length of 16 hours. Fungal infection was analyzed for mycelial growth by naked eye or microscopic observation 7 days after inoculation. Wild-type plants showed disease symptoms and died completely, while transgenic plants did not show any disease symptoms due to Phytophthora parasitica infection. The leaves of the control and tobacco transgenic tobacco were collected, stained with aniline blue, and observed under a fluorescent microscope. No hyphal growth was observed in the leaves of the transgenic plants, which showed bright fluorescence due to the presence of the autofluorescent substance in the sporangia.

[0033]

As described above in detail, the present invention provides a cDNA of an antimicrobial peptide derived from mature morning glory (Pharbitis nil L.), an expression vector of a recombinant plant containing the cDNA, and the use of the same. Provided is a method for producing a transgenic plant having antifungal resistance. Since the antimicrobial peptide Pn-AMP of the present invention has excellent antimicrobial activity against various plant pathogens, transgenic plants expressing the peptide have strong resistance to infection by various pathogens. Can have.

[0034] SEQUENCE LISTING <110> Korea Kumho Petrochemical Co., Ltd. <120> Transgenic Plants Expressing Antimicrobial Peptides derived from Seed of Pharbitis nil L. <130> PA99-224 <150> KR99-6622 <151> 1999-02 -27 <160> 9 <170> KOPATIN 1.0 <210> 1 <211> 41 <212> PRT <213> Pharbitis nil L. <400> 1 Gln Gln Cys Gly Ser Gln Ala Arg Gly Arg Leu Cys Gly Asn Gly Leu 1 5 10 15 Cys Cys Ser Gln Trp Gly Tyr Cys Gly Ser Thr Ala Ala Tla Cys Gly 20 25 30 Ala Gly Cys Gln Ser Gln Cys Lys Ser 35 40 <210> 2 <211> 40 <212> PRT <213> Pharbitis nil L. <400> 2 Gln Gln Cys Gly Arg Gln Ala Ser Gly Arg Leu Cys Gly Asn Gly Leu 1 5 10 15 Cys Cys Ser Gln Trp Gly Tyr Cys Gly Ser Thr Ala Ala Tyr Cys Gly 20 25 30 Ala Gly Cys Gln Ser Gln Cys Lys 35 40 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> synthetic primer based on amino acid sequence in chitin-binding do main of Pn-AMP1 and Pn-AMP2 <400> 3 tgytgywsnc artggggnta ytgycg 26 <210> 4 <211> 609 <212> DNA <213> Pharbitis nil L. <400> 4 aaa gaaagat caagaaacac ctactatact atacataaaa gagaaaatga aattctgtac 60 tatgtttctt gttgtcttgg ctttagccag cttgttgttg acaccatcaa caataatggc 120 acaacagtgt gggagtcaag cccgtgggcg tctgtgcggc aacggccttt gctgcagcca 180 gtggggctac tgtggctcca ctgcagccta ctgtggagct ggctgccaga gccaatgcaa 240 atctactgct gcttctgcca ccgacaccac caccactgca aaccaatcaa ccgctaagtc 300 ggatcccgcc ggcggtgcca actgatctca tcgatgatat gtacgttaga ccaccatgcg 360 ccgcacgccg atgtgccgta acctggccaa caaaaaataa aagatgtgtc gttgtttagt 420 ttaattctag ataggtaggg taggttatga aactaaagtg ggatggacat acatgtgatc 480 atctcacaaa tcaaatcttt ggatgctcct gcctactact ctatgtagtt tgtaatgtcc 540 gactactttg ttttaatttt gaataagatc gatgaggcgt gcatggttgc aaaaagctaa 600 gttccactt 609 <210> 5 <211> 279 <212> DNA <213> Pharbitis nil L. <400> 5 atgaaattct gtactatgtt tcttgttgtc ttggctttag ccagcttgtt gttgacacca 60 tcaacaataa tggcacaaca gtgtgggagt caagcccgtg ggcgtctgtg cggcaacggc 120 ctttgctgca gccagtgggg ctactgtggc tccactgcag cctactgtgg agctggctgc 180 cagagccaat gcaaatctac tgctgcttct gccaccgaca ccaccaccac tgcaaaccaa 240 tcaaccgcta agtcggatcc cgccggcggt gccaactga 279 <210> 6 <211> 605 <212> DNA <213> Pharbitis nil L. <400> 6 atatacacaa aagaattgaa aatgaaatac tgtactatgt ttattgttct cttgggttta 60 ggcagcttgt tgttgacacc aacaacaata atggcacaac agtgcgggag acaagccagt 120 gggcgtctgt gcggcaacgg cctttgctgc agccagtggg gctactgtgg ctccactgca 180 gcctactgtg gagctggttg ccagagccaa tgcaaatcta ctgctgcttc ttccaccacc 240 actaccactg caaaccaatc aaccgctaag tcggatcccg ccggcggtgc caactgatcg 300 atctcatcga taatgtatga ccacctccac catgcacgca cgcacgccga tatgccgtaa 360 cctgcaggct agcaaaaact aaaagatgtg tcgtcgttta ttttaattct agataggtag 420 gtaggttatg aaactaaagt gggatgaaca tccgtgtgat catctcacaa atcttagcta 480 ggatatgatg ctcctgccta ctactctatg tagtttgtaa tgtccgacta ctttatttta 540 attttgaata agatcaatgt ggcctgcatg gttgcaaaca ctaaaaaaaa aaaaaaaaaa 600 aaaaa 605 <210> 7 <211> 276 <212> DNA <213> Pharbitis nil L. <400> 7 atgaaatact gtactatgtt tattgttctc ttgggtttag gcagcttgtt gttgacacca 60 acaacaat aa tggcacaaca gtgcgggaga caagccagtg ggcgtctgtg cggcaacggc 120 ctttgctgca gccagtgggg ctactgtggc tccactgcag cctactgtgg agctggttgc 180 cagagccaat gcaaatctac tgctgcttct tccaccacca ctaccactgc aaaccaatca 240 accgctaagt cggatcccgc cggcggtgcc aactga 276 <210> 8 <211> 92 <212> PRT <213> Pharbitis nil L. <400> 8 Met Lys Phe Cys Thr Met Phe Leu Val Val Leu Ala Leu Ala Ser Leu 1 5 10 15 Leu Leu Thr Pro Ser Thr Ile Met Ala Gln Gln Cys Gly Ser Gln Ala 20 25 30 Arg Gly Arg Leu Cys Gly Asn Gly Leu Cys Cys Ser Gln Trp Gly Tyr 35 40 45 Cys Gly Ser Thr Ala Ala Tyr Cys Gly Ala Gly Cys Gln Ser Gln Cys 50 55 60 Lys Ser Thr Ala Ala Ser Ala Thr Asp Thr Thr Thr Thr Thr Ala Asn Gln 65 70 75 80 Ser Thr Ala Lys Ser Asp Pro Ala Gly Gly Ala Asn 85 90 <210> 9 <211> 91 <212> PRT <213> Pharbitis nil L. <400> 9 Met Lys Tyr Cys Thr Met Phe Ile Val Leu Leu Gly Leu Gly Ser Leu 1 5 10 15 Leu Leu Thr Pro Thr Thr Ile Met Ala Gln Gln Cys Gly Arg Gln Ala 20 25 30 Ser Gly Arg Leu Cys Gly Asn Gly Leu Cys Cys Ser Gln Trp Gly Tyr 35 4 0 45 Cys Gly Ser Thr Ala Ala Tyr Cys Gly Ala Gly Cys Gln Ser Gln Cys 50 55 60 Lys Ser Thr Ala Ala Ser Ser Thr Thr Thr Thr Thr Thr Ala Asn Gln Ser 65 70 75 80 Thr Ala Lys Ser Asp Pro Ala Gly Gly Ala Asn 85 90

[0035]

[Sequence List Free Text]

[0036]

[SEQ ID NO: 3] Primer synthesized based on amino acid sequence of chitin-binding domain of Pn-AMP1 and Pn-AMP2

[0037]

[Brief description of the drawings]

FIG. 1 is a drawing comparing the amino acid sequence of mature Pn-AMP1 (SEQ ID NO: 1) and the amino acid sequence of mature Pn-AMP2 (SEQ ID NO: 2) with the amino acid sequences of other chitin-binding proteins.

FIG. 2 is a genetic map of a recombinant plant expression vector pLES9806 containing Pn-AMP2 cDNA.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12N 1/21 // (C12N 1/21 C12R 1:01) (72) Inventor Rui Song Jun South Korea Gwangju Metropolitan City Gwangju-dong 756-2 Shindonga Apartment 102-906 (72) Inventor Jeong Chang-ho South Korea Republic of Korea Gwangju Metropolitan City Nishi-ku 572 Hanap-dong 100-1003 Modern Apartment

Claims (15)

[Claims] 1. An antimicrobial having the following amino acid sequence: QQCG-x-QA-y-GRLCGNGLCCSQWGYCGSTAAYCGAGCQSQCK-z (where x represents S or R, y represents R or S, and z is S or not). CDNA encoding a sex peptide. 2. An antimicrobial peptide Pn-AMP1 having an amino acid sequence of SEQ ID NO: 8 derived from morning glory (Pharbitis nil L.)
Represented by SEQ ID NO: 5 encoding the precursor protein of
cDNA. 3. A cDNA represented by SEQ ID NO: 7 encoding a precursor protein of antimicrobial peptide Pn-AMP2 having an amino acid sequence of SEQ ID NO: 9 derived from morning glory. 4. A plant expression vector containing the Pn-AMP1 cDNA of claim 2. 5. A plant expression vector pLES9806 comprising the Pn-AMP2 cDNA of claim 3 and represented by the genetic map of FIG. 6. The plant expression vector pLES9806 according to claim 5.
Agrobacterium tumefaciens transformed with Agrobacterium tumefaciens LBA4404 / pLES9806 (KCTC
0548BP). 7. A transgenic plant or progeny transformed with a gene encoding Pn-AMP1 and expressing said Pn-AMP1. 8. The transgenic plant or progeny thereof according to claim 7, wherein the transgenic plant is a tobacco or dicotyledonous plant. 9. A transgenic plant or progeny that is transformed with a gene encoding Pn-AMP2 and expresses the Pn-AMP2. 10. The transgenic plant or progeny thereof according to claim 9, wherein the transgenic plant is a tobacco or dicotyledonous plant. 11. A recombinant tobacco cell line expressing Pn-AMP2, Nicotiana tabacum cv.
Xanthi-nc / pLES9806 (KCTC 0549BP). 12. Using a recombinant microorganism transformed with an expression vector containing a gene encoding Pn-AMP as a vehicle,
Transgenic plant cells, wherein the plant cells express Pn-AMP; and culturing the plant cells under conditions that induce plant growth, producing a transgenic plant having antimicrobial resistance Method. 13. The method for producing a transgenic plant according to claim 12, wherein the recombinant microorganism is Agrobacterium tumefaciens LBA4404 (KCTC0548BP). 14. The method for producing a transgenic plant according to claim 12, wherein the plant is a tobacco or dicotyledonous plant. 15. Culture is carried out in a 25 ° C. incubator at day-length (day-lens).
gth) The method for producing a transgenic plant according to claim 12, which is performed in a nursery bed in which vermiculite, perlite, and peat moss are mixed at the same ratio under a condition of 16 hours.